The present invention is directed to a method for controlling the transbilayer distributions of ionizable lipids or ionizable proteins in a vesicle bilayer. More particularly, the invention describes methods for localizing ionizable lipids or ionizable proteins to either the inner or outer monolayer of unilamellar vesicles or to that of the outermost bilayer of multilamellar vesicles (MLV) in response to transmembrane ion gradients.
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (non-polar) "tails" of the lipid monolayers orient toward the center of the bilayer while the hydrophilic (polar) "heads" orient towards the aqueous phase.
The original liposome preparation of Bangham et al. (J. Mol. Biol., 13, 238-252 1965) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Then an appropriate amount of aqueous phase is added, the mixture is allowed to "swell", and the resulting liposome which consist of multilamellar vesicles (MLVs) are dispersed by mechanical means. This technique provides the basis for the development of the small sonicated unilamellar vesicles described by Papahadjopoulos et al. (Biochim. Biophys, Acta. 135, 624-638 1967), and large unilamellar vesicles.
Large unilamellar vesicles may be produced using an extrusion apparatus by a method described in Cullis et al., PCT 87/00238, Jan. 16, 1986, entitled "Extrusion Technique for Producing Unilamellar Vesicles," relevant portions of which are incorporated herein by reference. Vesicles made by this technique, LUVETS, are extruded under pressures of up to about 700 psi through a polycarbonate membrane filter. These vesicles may be exposed to at least one freeze and thaw cycle prior to the extrusion technique; this procedure is described in Bally et al., PCT 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies", relevant portions of which are incorporated herein by reference.
Another class of liposomes that may be used are those characterized as having substantially equal lamellar solute distribution. This class of liposomes is denominated as stable plurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 to Lenk, et al., monophasic vesicles as described in U.S. Pat. No. 4,558,579 to Fountain, et al., and frozen and thawed multilamellar vesicles (FATMLV) wherein the vesicles are exposed to at least one freeze and thaw cycle; this procedure is described in Bally et al., PCT Publication No. 87/00043, Jan. 15, 1987, entitled "Multilamellar Liposomes Having Improved Trapping Efficiencies" and incorporated herein by reference.
A variety of sterols and their water soluble derivatives have been used to form liposomes; see specifically Janoff et al., PCT 85/04578, Oct. 24, 1985, entitled "Steroidal Liposomes". Mayhew et al., WO 85/00968, published Mar. 14, 1985, describe a method for reducing the toxicity of drugs by encapsulating them in liposomes comprising alpha-tocopherol and certain derivates thereof. Also, a variety of tocopherols and their water soluble derivatives have been used to form liposomes, see Janoff et al., PCT 87/02219, Apr. 23, 1987, entitled "Alpha-Tocopherol-Based Vesicles" and incorporated herein by reference.
The method for preparing the sterol vesicles involves adding to an aqueous buffer a salt form of an organic acid derivative of a sterol capable of forming closed bilayers in an amount sufficient to form completely closed bilayers which entrap an aqueous compartment. A suspension of multilamellar vesicles is formed by shaking the mixture. The formation of vesicles is facilitated if the aqueous buffer also contains the counterion of the salt in solution.
The application of energy to the suspension, e.g., sonication, or extrusion of the vesicles through a French pressure cell (French Press) or through a filter of the appropriate pore size, will convert the multilamellar sterol vesicles to unilamellar vesicles.
The present invention provides a method for producing vesicles, preferably unilamellar vesicles, that exhibit transbilayer asymmetry of lipid in response to a transmembrane pH gradient. Alternatively, MLVs may also be made to exhibit this asymmetry in their outermost bilayer.
There is an ongoing need for liposomes having enhanced pharmacokinetics, sustained release characteristics, stability and targeting ability.